Homogenous protein assays, despite the potential for mix-and-read workflows, have eluded widespread acceptance due to interferences in biological matrices and limited multiplexability. Here, we employ standard qPCR instrumentation for thermofluorimetric analysis of bivalent probe (TFAB) assemblies, allowing protein levels to be quantitatively translated into multiplexable DNA melting transitions within 30 min. As protein-bound bivalent probes are thermodynamically more stable than unbound probes, differential thermal analysis can remove background analytically, without physical separation. Using either antibody-oligonucleotides or aptamers as probes, TFAB is validated for protein quantification in buffer, human serum, and human plasma and for assaying hormone secretions from endocrine cells. The direct optical method exhibits superior scalability, allowing detection of only 1 amol of protein in microfluidic channels of 100 pL volume. Overall, we demonstrate TFAB as a robust and generalizable homogeneous protein assay with superior performance in biological matrices.

Employing 3D-printed templates for macro-to-micro interfacing, a passively operated polydimethysiloxane (PDMS) microfluidic device was designed for time-resolved secretion sampling from primary murine islets and epidiymal white adipose tissue explants. Interfacing in similar devices is typically accomplished through manually punched or drilled fluidic reservoirs. We previously introduced the concept of using hand fabricated polymer inserts to template cell culture and sampling reservoirs into PDMS devices, allowing rapid stimulation and sampling of endocrine tissue. However, fabrication of the fluidic reservoirs was time consuming, tedious, and was prone to errors during device curing. Here, we have implemented computer-aided design and 3D printing to circumvent these fabrication obstacles. In addition to rapid prototyping and design iteration advantages, the ability to match these 3D-printed interface templates with channel patterns is highly beneficial. By digitizing the template fabrication process, more robust components can be produced with reduced fabrication variability. Herein, 3D-printed templates were used for sculpting millimetre-scale reservoirs into the above-channel, bulk PDMS in passively-operated, eight-channel devices designed for time-resolved secretion sampling of murine tissue. Devices were proven functional by temporally assaying glucose-stimulated insulin secretion from <10 pancreatic islets and glycerol secretion from 2-mm adipose tissue explants, suggesting that 3D-printed interface templates could be applicable to a variety of cells and tissue types. More generally, this work validates desktop 3D printers as versatile interfacing tools in microfluidic laboratories.

Electrochemical bioanalytical sensors with oligonucleotide transducer molecules have been recently extended for quantifying a wide range of biomolecules, from small drugs to large proteins. Short DNA or RNA strands have gained attention recently due to the existence of circulating oligonucleotides in human blood, yet challenges remain for adequately sensing these targets at electrode surfaces. In this work, we have developed a quantitative electrochemical method which uses target-induced proximity of a single-branched DNA structure to drive hybridization at an electrode surface, with readout by square-wave voltammetry (SWV). Using custom instrumentation, we first show that precise control of temperature can provide both electrochemical signal amplification and background signal depreciation in SWV readout of small oligonucleotides. Next, we thoroughly compared 25 different combinations of binding energies by their signal-to-background ratios and differences. These data served as a guide to select the optimal parameters of binding energy, SWV frequency, and assay temperature. Finally, the influence of experimental workflow on the sensitivity and limit of detection (LOD) of the sensor is demonstrated. This study highlights the importance of precisely controlling temperature and SWV frequency in DNA-driven assays on electrode surfaces while also presenting a novel instrumental design for fine-tuning of such systems.

One of the key factors limiting sensitivity in many electrochemical assays is the nonfaradaic or capacitive current. This is particularly true in modern assay systems based on DNA monolayers at gold electrode surfaces, which have shown great promise for bioanalysis in complex milieu such as whole blood or serum. While various changes in analytical parameters, redox reporter molecules, DNA structures, probe coverage, and electrode surface area have been shown useful, background reduction by hardware subtraction has not yet been explored for these assays. Here, we introduce new electrochemistry hardware that considerably suppresses nonfaradaic currents through real-time analog subtraction during current-to-voltage conversion in the potentiostat. This differential potentiostat (DiffStat) configuration is shown to suppress or remove capacitance currents in chronoamperometry, cyclic voltammetry, and square-wave voltammetry measurements applied to nucleic acid hybridization assays at the electrode surface. The DiffStat makes larger electrodes and higher sensitivity settings accessible to the user, providing order-of-magnitude improvements in sensitivity, and it also significantly simplifies data processing to extract faradaic currents in square-wave voltammetry (SWV). Because two working electrodes are used for differential measurements, unique arrangements are introduced such as converting signal-OFF assays to signal-ON assays or background drift correction in 50% human serum. Overall, this new potentiostat design should be helpful not only in improving the sensitivity of most electrochemical assays, but it should also better support adaptation of assays to the point-of-care by circumventing complex data processing.